Re: IEF efficiency

Dan Crimmins (crimmins@pathbox.wustl.edu)
Mon, 23 Feb 1998 08:50:20 -0600 (CST)

At 12:33 PM 2/23/98 +0100, Amanda Hall wrote:
>Hi everyone,
>
>Back in January Judith Airey asked a question about efficiency of IEF gels.
>I am having a similar problem and I missed the responses so I hope you
>don't mind me asking a similar question. I am a beginner with 2D's and
>would appeciate any help.
>
>As with Judith I am finding that the amount of protein present at the end
>of the 2nd dimension run is much less than expected. I am using the
>Pharmacia IPG strips for the 1st dimension run.
>
>My sample is a mixture of membrane proteins in CHAPS detergent. The sample
>has protease inhibitors present so proteolysis shouldn't play a major part.
>Could the solubility of the membrane proteins be the problem, perhaps with
>the protein precipitating out during the 1st dimension run? If this is a
>potential problem has anyone got any useful suggestions for increasing the
>solubility of the sample, or any ways to improve efficiency generally.
>
>Any comments would be gratefully received
>
>Thank you very much
>
>Amanda HAll
>
>
>Amanda Hall
>Professional Officer
>Newcastle Protein
>University of Newcastle
>
>ph 02 4921 7299
>email: mbah@medicine.newcastle.edu.au
>
>
>
>

Amanda,

See Rabilloud, T. (1996) Electrophoresis 17, 813-829.
Solubilization of proteins for electrophoretic analysis, and

Pasquali, C. et al., (1997) Electrophoresis 18, 2573-2581.
Preparative two-dimensional electrophoresis of membrane proteins.

Best regards,

Dan L. Crimmins
Washington University School of Medicine
Dept. Pathology/Division of Laboratory Medicine
660 S. Euclid Ave., Box 8118
St. Louis, MO 63110
Phone: 314-454-8514; Fax: 314-362-1461
e-mail: crimmins@labmed.wustl.edu